Total-EGFR L858R assay principle

The HTRF Total-EGFR L858R assay quantifies the expression level of mutated EGFR L858R in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-EGFR Mutant L858R assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of EGFR Mutant L858R in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-EGFR L858R two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total-EGFR L858R HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored

Total-EGFR L858R one-plate assay protocol

Detection of Total-EGFR Mutant L858R with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Total EGFR L858R assay validated in relevant EGFR mutated cell lines.

Total EGFR L858R expression levels were assessed with the HTRF Total EGFR DEL19 kit in different cell lines: H1975 and H3255 cells (expressing EGFR L858R mutant), A431 and A549 cells (expressing wild type EGFR), and H1650 and HCC827 (expressing the EGFR DEL19 mutant).

The different cell lines were cultured in T175 flasks in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking. 16 µL of each lysate diluted by 2 were transferred into a low volume white microplate before the addition of 4 µL of HTRF total EGFR L858R detection reagents.

As expected, no signal was detected in the H1650, HCC827, A431, and A549 cell lines, demonstrating the specificity of the HTRF Total EGFR L858R kit to detect mutated EGFR L858R only.

HTRF Total EGFR L858R assay compared to Western Blot

H1975 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total EGFR L858R detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

In these conditions, the HTRF total EGFR L858R assay is 17 times more sensitive than the Western Blot technique.

Total EGFR Signaling Pathway

EGFR is a receptor tyrosine kinase that belongs to the ErbB family. Binding of EGFR ligands drives receptor homodimerization or hetero-dimerization, leading to the activation of the EGFR tyrosine kinase domain and specific tyrosine residues.

The phosphorylated tyrosine residues then provide docking sites for a variety of factors that induce downstream activation of several signal transduction cascades.

The signals transmitted from the EGF receptor to the nucleus lead to the regulation of various biological functions, such as cell proliferation, differentiation, survival, adhesion, migration, and angiogenesis.

Mutations that lead to overexpression or hyperactivity of EGFR are associated with a number of cancers, making EGFR a key target for anti-cancer therapies.